Comparison of the Proliferation and Apoptosis of NIH 3T3 cells Treated with DJ-1~(L166P) or DJ-1~(M26I)

Objective To explore the relationship between DJ-1,DJ-1M26I,DJ-1L166P with the cell proliferation and apoptosis of NIH 3T3 cells at cellular level,and provide a basis for the construction of a transgenic animal model of Parkinson's disease and further study on the pathogenesis of this disease.Methods Recombinant plasmids pcDNA3.1/myc-His-DJ-1,pcDNA3.1/myc-His-DJ-1L166P and pcDNA3.1/myc-His-DJ-1M26I were transfected into NIH 3T3 cells,respectively,using lipofectamine.The cells were screened with G418 at a dose of 500 μg/mL.Stable clones were identified on the DNA,RNA and protein levels.MTT assay and annexin V-FITC kit were used to detect the viability and apoptosis of those stable cell clones.Results After the G418-screening of of NIH 3T3 cells transfected with recombinant plasmids pcDNA3.1/myc-His-DJ-1,pcDNA3.1/myc-His-DJ-1L166P or pcDNA3.1/myc-His-DJ-1M26I,one,four and three positive clones were obtained,respectively,by PCR detection.RT-PCR and Western blot detected the expression of DJ-1-His in the positive clones.NIH 3T3 cells transfected with DJ-1L166P and DJ-1M26I had a higher expression of caspase-3 mRNA than normal NIH 3T3 cells,while NIH3T3 cells transfected with DJ-1 had a lower expression.MTT assay showed that NIH 3T3 positive cells transfected with DJ-1L166P and DJ-1M26I had a lower proliferation rate than that of normal NIH3T3 cells(P0.05),while the NIH 3T3 positive cells carrying DJ-1 gene did not show significant difference compared with the normal NIH 3T3 cells.Apoptosis test indicated that the apoptosis rates of DJ-1L166P and DJ-1M26I transfected cells were higher than that of normal NIH 3T3 cells,however the apoptosis rate of the DJ-1-transfected cells was significantly lower than that of normal NIH 3T3 cells(P0.05).Conclusions DJ-1L166P and DJ-1M26I mutations reduce the proliferation of NIH 3T3 cells.DJ-1L166P and DJ-1M26I mutations also enhance apoptosis in NIH 3T3 cells.Their effects on NIH 3T3 cell proliferation and apoptosis are similar.